The enzymes of the bc1 complex family (ubihydroquinone:cytochrome c (or c2) oxidoreductases, and the closely related b6f complexes of oxygenic photosynthesis), carry the energy flux of the biosphere, serving as the central enzymes of respiratory and photosynthetic electron transfer chains. The aim of the parent project has been to understand how these important enzymes function. Structures for several mitochondrial complexes have recently provided new insights on function that have led us to suggest some novel extensions of the basic Q-cycle mechanism. These have included a dramatic movement of the extrinsic domain of the iron-sulfur protein between two reaction interfaces, and a revised mechanism for the reaction by which quinol is oxidized, with specific evidence for the nature of the enzyme-substrate complex, and the pathways for release of both electrons and protons. The structures have also provided a more detailed understanding of the quinone reduction site, for which we have suggested a novel mechanism. Apart from its intrinsic interest, the bc1 complex is a major site of production of oxygen radicals, which cause cell aging and DNA damage leading to cancer. Production of superoxide anion by the bc1 complex is mechanistically linked to quinol oxidation, but evolution has minimized this suicidal side-reaction. Our studies will provide an understanding of this medically important process. In this FIRCA proposal, we will make use of the structures in an extended exploration of the local context of molecular mechanism, using high-resolution EPR spectroscopic methods, and taking advantage of the biophysical, molecular engineering and biochemical protocols developed under the parent grant. We will use 2D ESEEM to study the interaction of reaction intermediates (semiquinone and reduced iron-sulfur protein) with endogenous nuclear spins of the neighboring protein, and synthesize ubiquinones isotopically labeled at specific positions with nuclear spins, to explore the interaction of these with paramagnetic species generated during catalytic turnover. Synthetic aspects of this research will be done primarily in Russia as an extension of NIH grant 2 R0l GM 35438-13.